Neuroglia Infection by Rabies Virus After Anterograde Virus Spread in Peripheral Neurons

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bioRxiv preprint doi: https://doi.org/10.1101/2020.09.20.305078; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 Neuroglia Infection by Rabies Virus after Anterograde Virus Spread in Peripheral Neurons

3 Madlin Potratz, Luca M. Zaeck, Carlotta Weigel, Antonia Klein, Conrad M. Freuling, Thomas Müller, Stefan

4 Finke*

5 Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Institute of Molecular Virology

6 and Cell Biology, 17493 Greifswald-Insel Riems, Germany

10 * Correspondence: [email protected]; Tel.: +49-38351-71283; Fax.: +49-38351-71151

16 Keywords: rabies pathology, tissue optical clearing, 3D tissue imaging, light sheet microscopy, neuroglia

17 infection, Schwann cell

19 Abstract

20 The highly neurotropic rabies virus (RABV) enters peripheral neurons at axon termini and requires long distance

21 axonal transport and trans-synaptic spread between neurons for the infection of the central nervous system (CNS).

22 Whereas laboratory strains are almost exclusively detected in neurons, recent 3D imaging of field RABV-infected

23 brains revealed an remarkably high proportion of infected astroglia, indicating that in contrast to attenuated lab

24 strains highly virulent field viruses are able to suppress astrocyte mediated innate immune responses and virus

25 elimination pathways. While fundamental for CNS invasion, in vivo field RABV spread and tropism in peripheral

26 tissues is understudied. Here, we used three-dimensional light sheet and confocal laser scanning microscopy to

27 investigate the in vivo distribution patterns of a field RABV clone in cleared high-volume tissue samples after

28 infection via a natural (intramuscular; hind leg) and an artificial (intracranial) inoculation route. Immunostaining

29 of virus and host markers provided a comprehensive overview of RABV infection in the CNS and peripheral

30 nerves after centripetal and centrifugal virus spread. Importantly, we identified non-neuronal, axon-ensheathing

31 neuroglia (Schwann cells, SCs) in peripheral nerves of the hind leg and facial regions as a target cell population

32 of field RABV. This suggest that virus release from axons and infected SCs is part of the RABV in vivo cycle and

33 may affect RABV-related demyelination of peripheral neurons and local innate immune responses. Detection of

34 RABV in axon surrounding myelinating SCs after i.c. infection further provided evidence for anterograde spread

35 of RABV, highlighting that RABV axonal transport and spread of infectious virus in peripheral nerves is not

36 exclusively retrograde. Our data support a new model in which, comparable to CNS neuroglia, SC infection in

37 peripheral nerves suppresses glia-mediated innate immunity and delays antiviral host responses required for

38 successful transport from the peripheral infection sites to the brain.

41 Introduction

42 Rabies is estimated to cause 59.000 human deaths annually in over 150 countries, with 95% of cases occurring in

43 Africa and Asia (reviewed in [26,21]). The disease is caused by a plethora of highly neurotropic, non-segmented,

44 single-stranded RNA viruses of negative polarity belonging to the family Rhabdoviridae of the order

45 Mononegavirales, of which the rabies virus (RABV) is the type species of the Lyssavirus genus [2]. RABV and

46 other lyssaviruses enter the nervous system at the synapses of peripheral neurons. After receptor-mediated

47 endocytosis, the virions remain in endosomal vesicles and hijack the retrograde axonal transport machinery

48 [33,23,59] to reach the neuron soma, where virus ribonucleoproteins (RNPs) are released into the cytoplasm [47]

49 for virus gene expression and replication. Newly formed virus is then transported to post-synaptic membranes,

50 followed by trans-synaptic spread to connected neurons [12]. Prolonged replication of pathogenic RABV in the

51 brain eventually leads to the onset of a progressive rabies encephalitis, invariably resulting in the fatal disease

52 progression. In animals suffering from RABV encephalitis, the virus can be detected in multiple nervous or

53 innervated tissues including hind leg, spinal cord, brain, face, and salivary glands [5,15]. For the transmission of

54 infectious virus to new hosts via virus-containing saliva, centrifugal spread of RABV in the peripheral nervous

55 system (PNS) is required [18]. The secretion of RABV-containing saliva by salivary glands is commonly accepted

56 as the main source of virus shedding and virus transmission to uninfected animals via bites (reviewed in [21,16]).

57 Detection of RABV in sensory neurons of the olfactory epithelium and other tissues during late stages of infection

58 indicates that, at least in these late phases of disease, the axonal transport is not exclusively retrograde (reviewed

59 in [16]). In fact, studies have shown that RABV enters the anterograde axonal transport pathway in cultivated

60 primary rat dorsal root ganglion (DRG) neurons [6] and in mice [70]. However, the consequences of anterograde

61 transport of RABV is still controversially discussed, even though infection through intradermal sensory neurons

62 represents a likely route of entry for lyssaviruses as a result from bat bites [7].

63 Because of their trans-synaptic spread, RABVs constitute ideal viruses to map neuronal connections [67,66] as

64 even attenuated RABVs enter axonal transport pathways [33]. More neuroinvasive yet still lab-adapted strains like

65 CVS-11 have been used as polysynaptic neuron tracers and the resultant insights have subsequently been used to

66 explain in vivo RABV spread and pathogenesis [61]. However, already between different CVS strains, substantial

67 differences in pathogenicity exist [39], raising the question whether knowledge from established lab-adapted

68 strains can be extrapolated to explain actual field RABV infection and pathogenesis.

69 The advent of efficient full-genome cloning techniques opened novel avenues for the analysis of virus variability

70 in combination with phenotypical characterization of recombinant field viruses at a clonal level [43]. For example,

71 a recent comparison of field and lab RABV clones in mice revealed that field viruses not only showed a higher 3

72 virulence compared to lab strains such as CVS-11 but also substantially differ in their ability to establish infection

73 in non-neuronal astroglia in the central nervous system (CNS) [48]. In contrast to attenuated lab-adapted RABV,

74 which is supposed to be eliminated from infected glial cells (astrocytes) by type I interferon-induced immune

75 reactions [46], it has been hypothesized that rapid onset of field virus gene expression in non-neuronal astrocytes

76 blocks inhibitory innate immune activation and responses [48]. This may not only affect RABV replication in glial

77 cells directly but also in surrounding neurons via paracrine signalling. Thus, this mechanism may be crucial to

78 RABV spread and pathogenesis in vivo. Apart from astrocytes, in vivo infection of non-neuronal muscle,

79 epidermal, and epithelial cells have been described [12,5,13,42]. In foxes, which had been orally immunized with

80 live-attenuated RABV, infected cells can be detected in the epithelium of lymphoid tonsils [63,56]. In vitro, even

81 immature DCs or monocytes can be infected [35]. Overall, knowledge on the broad spectrum infection pattern of

82 RABV in vivo, particularly of field RABV strains, is still incomplete and the identification of PNS and non-

83 neuronal cells involved in RABV spread and pathogenesis has been given little attention.

84 Therefore, by using a highly virulent field RABV clone of dog origin (rRABV Dog) [43], we set out to

85 systematically and comprehensively elucidate the in vivo spread and cell tropism of a field RABV strain. So far,

86 technical limitations in conventional histology to visualize infection processes in complex tissues in large three-

87 dimensional (3D) volumes at a mesoscopic scale and high resolution hindered a comprehensive overview of RABV

88 cell tropism, in vivo spread, and pathogenesis. Immunofluorescence-compatible tissue clearing iDISCO and

89 uDISCO (immunolabeling-enabled and ultimate three-dimensional imaging of solvent-cleared organs,

90 respectively) protocols [45,49] recently allowed us to perform high-resolutions 3D immunofluorescence analyses

91 of thick RABV-infected tissues slices [69,48].

92 Here, we investigated the in vivo spread of a highly virulent field RABV clone (rRABV Dog) through the CNS

93 and PNS after intramuscular (i.m.) and intracranial (i.c.) inoculation. Using 3D immunofluorescence imaging of

94 solvent-cleared tissues by light sheet and confocal laser scanning microscopy, we identified RABV infections in

95 hind leg nerves, spinal cord, brain, and nerves of the facial region. These data provide a broad overview of field

96 RABV distribution in the clinical phase of rabies encephalitis. Notably, we were able to identify Schwann cells

97 (SCs) as a so far unrecognized non-neuronal RABV target cell population, which may be involved in regulating

98 RABV infection and, thus, pathogenesis in peripheral nerve tissues. Moreover, infection of SCs after centrifugal

99 RABV spread provides evidence for anterograde axonal transport and infectious virus release at distal axon

100 membrane sites of peripheral neurons.

101

102 4

103 Materials and Methods

104 Viruses. The recombinant field virus clone rRABV Dog [43] was derived from an isolate originating from an

105 infected dog from Azerbaijan, which was used in a challenge study [64]. RABV Bat (FLI virus archive ID N

106 13240) was isolated from an insectivorous North American bat.

107 Animal trials. Six- to eight-week-old BALB/c mice (Charles-River, Germany) were i.m.-infected with decreasing

108 doses of rRABV Dog. Four groups of six animals each were anesthetized with isoflurane and infected with 3x100,

1 2 3 109 3x10 , 3x10 and 3x10 TCID50/mouse by injection of 30 µL of virus suspension in the femoral hind leg muscle.

2 110 Additionally, three mice were i.c.-infected at a dose of 1x10 TCID50/mouse. Body weight and clinical scores from

111 one to five (1: ruffled fur, hunched back; 2: slowed movement, circular motions, weight loss > 15%; 3: tremors,

112 shaky movement, seizures, weight loss > 20%; 4: paralysis, weight loss > 25%; 5: coma/death) were determined

113 daily until day 21 post-infection or day of euthanasia. After reaching a clinical score of two, the animals were

114 anaesthetized with isoflurane and euthanized by decapitation. Samples were taken, fixed with 4%

115 paraformaldehyde (PFA) for one week, and stored for further processing. All remaining animals were euthanized

116 at 21 dpi. The animal approaches were evaluated by the responsible animal care, use, and ethics committee of the

117 State Office for Agriculture, Food Safety, and Fishery in Mecklenburg-Western Pomerania (LALFF M-V) and

118 gained approval with permission 7221.3-2-047/19). For RABV Bat, archived tissue samples (hind legs, spinal

119 columns, and heads) from previous virus characterization experiments were used (LALFF M-V permission

120 7221.3-2.1-001/18).

121 Detection of RABV RNA in oral swabs and salivary glands via RT-PCR. Salivary gland samples were

122 homogenized in 1 ml cell culture medium in the UPHO Ultimate Homogenizer (Geneye, Hong Kong) using a 3

123 mm steal bead. Oral swab samples were transferred into 1 ml of culture medium. Total RNA from oropharyngeal

124 swabs as well as from salivary gland tissues were extracted using the NucleoMagVet kit (Macherey&Nagel,

125 Germany) according to manufacturer’s instructions in a KingFisher/BioSprint 96 magnetic particle processor

126 (Qiagen, Germany). RABV RNA was detected by the R14-assay, a RT-qPCR targeting the N-gene [27]. The RT-

127 qPCR reaction was prepared using the AgPath-ID-One-Step RT-PCR kit (Thermo Fisher Scientific, USA) adjusted

128 to a volume of 12.5 µl [19]. RT-qPCRs were performed on a BioRad real-time CFX96 detection system (Bio-Rad,

129 USA).

130 Antibodies and dyes. To detect RABV in the tissues, a polyclonal rabbit serum against recombinant RABV P

131 protein (P160-5; 1:3,000 in PTwH [0.2% Tween 20 in PBS with 10 µg/mL heparin]) was used [44]. The following

132 first antibodies and dyes were obtained from the respective suppliers: chicken anti-NEFM (Thermo Fisher, USA;

133 #PA1-16758, RRID:AB_2282551; 1:1,000 in PTwH), guinea pig anti-NEFM (Synaptic Systems, Germany; 5

134 #171204, RRID:AB_2619872; 1:400 in PTwH), chicken anti-MBP (Thermo Fisher; #PA1-10008,

135 RRID:AB_1077024; 1:500 in PTwH), TO-PROTM-3 iodide (Thermo Fisher; #T3605; 1:1,000 in PTwH.) As

136 secondary antibodies donkey anti-rabbit Alexa Fluor® 568 (Thermo Fisher; #A10042, RRID:AB_2534017),

137 donkey anti-chicken Alexa Fluor® 488 (Dianova, Germany; #703-545-155, RRID:AB_2340375), donkey anti-

138 guinea pig Alexa Fluor® 647 (Dianova; #706-605-148, RRID:AB_10895029) were used, each at dilution of 1:500

139 in PTwH.

140 iDISCO-based immunostaining and uDISCO-based clearing of tissue samples. Staining and clearing

141 protocols were adapted from earlier reports [45,49] and were performed as described previously [69,48]. Hind legs

142 were skinned prior to fixation. Prior to the slicing, PFA-fixed hard tissues, which included hind legs, spinal

143 columns, and heads, were decalcified with 20% ethylenediaminetetraacetic acid (EDTA) [w/v] in H2Odd (pH was

144 adjusted to 7.0) for four days at 37 °C. The EDTA solution was exchanged daily. Afterwards the decalcified tissues

145 were cut into 1 mm thick sections using a scalpel. For brains, as soft tissues, a vibratome (VT1200S, Leica

146 Biosystems, Germany) was used. After treatment with increasing methanol concentrations in distilled water (20%,

147 40%, 60%, 80%, and twice 100%) and bleaching with 5% H2O2 in pure methanol at 4 °C, the samples were

148 rehydrated with decreasing concentrations of methanol in distilled water (80%, 60%, 40%, and 20%). After

149 washing with PBS and 0.2% Triton X-100 in PBS, the samples were permeabilized for 2 days at

150 37 °C in 0.2% Triton X-100/20% DMSO/0.3 M glycine in PBS. After subsequent blocking with 6% donkey

151 serum/0.2% Triton X-100/10% DMSO in PBS at 37 °C for another 48 h, primary antibodies in PTwH were added

152 for 5 days with refreshment of the antibody solution after 2.5 days. The samples were washed with PTwH four

153 times until the next day, when secondary antibodies in PTwH were added for 5 days with refreshment of the

154 antibody solution after 2.5 days. After further washing for four times until the next day, the tissues were dehydrated

155 by a series of tert-butanol (TBA) solutions (from 30% to 96% in distilled water), each step for 2 h (except over

156 night incubation with for 80% TBA), final incubation in 100% TBA and subsequent clearing in BABB-D15 with

157 0.4 vol% DL-α-tocopherol until they were optically transparent (2 – 6 h). BABB-D15 is a combination of benzyl

158 alcohol (BA) and benzyl benzoate (BB) at a ratio of 1:2, which is mixed at a ratio of 15:1 with diphenyl ether

159 (DPE). The samples were either analyzed directly by light sheet microscopy or were transferred to 3D-printed

160 imaging chambers as described before (Zaeck et al., 2019.)

161 Light sheet and confocal laser scanning microscopy. For light sheet microscopy, a LaVision UltraMicroscope

162 II with an Andor Zyla 5.5 sCMOS Camera, an Olympus MVX-10 Zoom Body (magnification range: 0.63 – 6.3x),

163 and an Olympus MVPLAPO 2x objective (NA = 0.5) equipped with a dipping cap was used. Z-stacks were

164 acquired with a light sheet NA of 0.156, a light sheet width of 100%, and a z-step size of 2 µm. LaVision BioTec

165 ImSpector Software (v7.0.127.0) was used for image acquisition.

166 Confocal laser scanning microscopy was performed with a Leica DMI 6000 TCS SP5 equipped with a HC PL

167 APO CS2 40x water immersion objective (NA = 1.1; Leica, Germany; #15506360), using the Leica Application

168 Suite Advanced Fluorescence software (V. 2.7.3.9723) and a z-step size of 0.5 µm.

169 Image processing. For image processing, a Dell Precision 7920 workstation was used (CPU: Intel Xeon Gold

170 5118, GPU: Nvidia Quadro P5000, RAM: 128 GB 2666 MHz DDR4, SSD: 2 TB). Acquired image stacks were

171 processed using the ImageJ (v1.52h) distribution package Fiji [53,54] or arivis Vision4D (arivis, Germany; v3.2.0).

172 Brightness and contrast were adjusted for each channel and, if necessary, channels were denoised. Either

173 maximum-z-projections or volumetric 3D-projections are shown.

174

175 Results

176 Field virus clone rRABV Dog undergoes the complete RABV infection cycle in adult BALC/c mice. Mouse

177 models differ in their age-dependent susceptibility to fixed RABV lab strains and field isolates [11]. To confirm

178 the field virus character of rRABV Dog and its suitability for investigation of the complete RABV in vivo cycle

179 from infected muscle at the site of entry to secretion in saliva in adult mice, six- to eight-week-old BALB/c mice

180 were inoculated by the i.m. route (hind leg muscle) with decreasing infectious virus doses. Although mortality was

181 dose-dependent, even very low doses (30 TCID50/mouse) of rRABV Dog were sufficient to cause rabies disease

182 and release of infectious virus in nearly all inoculated mice, thus, highlighting the high virulence of this field virus

183 RABV clone. Comparable to field RABV isolates, even at an extremely low infection dose (3 TCID50/mouse), the

184 complete in vivo cycle had been undergone by animals that succumbed to infection, as shown by detection of viral

185 RNA in both salivary glands and in oral swabs using RT-qPCR (Table 1; Fig. S1).

186 Table 1: Dose-dependent onset of clinical rabies signs after i.m. application of rRABV Dog in the upper

187 hind leg muscle of six- to eight-week old BALB/c mice. Salivary gland and oral swabs were virus RNA positive

188 even at low infection doses. *One oral swab not tested.

virus dose euthanized salivary gland oral swab

TCID50/mouse (humane endpoint reached) virus RNA virus RNA

3,000 6/6 6/6 5*/6

300 5/6 5/6 5/6

30 5/6 5/6 5/6

3 2/6 2/6 2/6

189

190 RABV infects peripheral neuron-associated neuroglia in the hind leg after i.m. inoculation. Immunostaining

191 of solvent-cleared hind leg slices of diseased animals for RABV phosphoprotein (P), neurofilament M (NEFM),

192 and cell nuclei (TO-PROTM-3) was performed for light sheet and confocal laser scanning microscopy analysis (Fig.

193 1). RABV infection of peripheral nerves innervating the femoral tissue could be demonstrated (Fig. 1 A-C, red).

194 Reconstruction of a 3D femoral tissue volume allowed visualization of the distribution of infected nerve fibres in

195 their spatial environment (Fig. 1B, Video S1). The specificity of the RABV immunofluorescence was confirmed

196 by using non-infected hind leg tissues as negative controls (Fig. S3). At a higher resolution, axons were identified

197 via staining by the neuronal cytoskeleton marker (NEFM) (Fig. 1D,E, green) but, notably, did not co-localize with

198 RABV P (Fig. 1E). As RABV-infected peripheral nerves could be visualized in all serial femoral slices analysed

199 (Fig. 1F-J), our approach proved to be appropriate to reconstruct a 3D model of RABV infection patterns in

200 peripheral tissues.

201 Further confocal laser scanning analysis confirmed the lack of co-localization of RABV P with NEFM in axons

202 (Fig. 2A,B). Rather, RABV P specific signals were arranged next to or around the tubular NEFM signals (Fig.

203 2C,D, arrows; Video S2). Similar spatial arrangement patterns were observed after staining for the Schwann cell

204 (SC) marker myelin basic protein (MBP) (Fig. 2E-K). MBP is known to be part of the myelin sheath of SCs that

205 forms tubular structures around the axons (reviewed in [51]) and accordingly, appeared as hollow tubules that

206 were surrounded by RABV P (Fig. 2 G-K, arrowheads; Video S3). Corroborated by the presence of nuclei in

207 RABV-infected cells (Fig. 2K), this suggests that RABV P accumulated in the cytoplasm of axon-ensheathing

208 SCs. This provides evidence for the infection of and virus gene expression in neuroglia of the PNS in the clinical

209 phase of RABV encephalitis after i.m. infection.

210 Infection of Schwann cells after i.c. inoculation strongly supports anterograde spread of RABV into the

211 periphery. Late phase infection of myelinating SCs in the PNS of i.m.-infected mice could be a result of unspecific

212 virus release from abundantly and long-term RABV-infected myelinated neurons. To investigate, whether RABV

213 also enters the PNS after centrifugal spread from the brain and whether this also leads to infection of myelinating

214 SCs, femoral tissue from i.c.-infected mice was analyzed.

215 Detection of RABV antigen in the PNS revealed that innervating nerves in femoral tissue can get infected after

216 centrifugal spread of the virus from the brain (Fig. 3A-G). As observed for i.m. inoculations, RABV P fluorescence

217 signals surrounded rather than overlapped with NEFM-positive axons (Fig. 3D,E). Staining for RABV P, MBP,

218 and NEFM (Fig. 3F-K) revealed a three-layered structure with RABV P-specific signals surrounding the MBP-

219 sheath of axons (Fig. 3H-K, Video S4). Infection of myelinated SCs after i.c. inoculation demonstrated that

220 infectious virus was released from peripheral axons, infecting the surrounding axon-associated, non-neuronal cells.

221 RABV exhibits a strong, infection route-independent infection of the spinal cord. To assess the degree of

222 infection of the spinal cord by field virus clone rRABV Dog after different routes of infection, spinal column slices

223 (Fig. S2C) of i.m.- and i.c.-infected mice were systematically analyzed. Independent of the inoculation route,

224 multiple neurons in the spinal cord and neurons projecting out of the same were positive for RABV P (Fig. 4).

225 Higher magnification image stacks of the spinal cord revealed RABV P staining to be primarily localized in the

226 grey matter, surrounded by NEFM-positive white matter (Fig. 4C,I, star and triangle, respectively). Indeed, white

227 and grey matter (Fig. 4D,J, star and triangle, respectively) revealed multiple NEFM-positive axons of which some

228 were infected (Fig. 4E,K). In contrast to the peripheral femoral axons, where the RABV P staining almost

229 exclusively surrounded the tubular NEFM structures (Fig. 2C,D and 3D,E), here, RABV P- and NEFM-specific 9

230 signals perfectly co-localized (Fig. E,K), suggesting that the axons contained high levels of RABV P protein.

231 Peripheral nerves projecting from the spinal cord also revealed double positive axons (Fig. 4F-H, L-N). However,

232 also RABV P not overlapping with NEFM was observed (Fig. 4H, arrow). In some projecting axon bundles (Fig.

233 4L), almost all axons were RABV-positive as indicated by co-localization of RABV P and NEFM (Fig. 4M,N).

234 Pronounced infection of various tissues and sensory neurons of the facial head region. Since centrifugal

235 spread to the salivary glands is considered an inevitable step in RABV transmission to other hosts, the extent of

236 RABV infection in tissues of the facial head regions was investigated by imaging of coronal head slices (Fig. S2B).

237 Except for the olfactory bulb, the brain was removed prior to sectioning for separate processing. 3D volume

238 reconstruction from light sheet microscopy z-stacks demonstrated abundant RABV P specific signals throughout

239 various areas of the coronal head slice both after i.c. (Fig. 5A,B; Video S5) and i.m. inoculation (Fig. 6A,B, Fig.

240 S4; Video S6). RABV-infected cells could be identified in the orbital (Fig. 5C,D and 6C,D), nasal (Fig. 5E,F and

241 6E,F), and oral cavity (Fig. 5G-J and 6G-J). Morphologies of the infected cells revealed a structure typical for

242 sensory neurons in the olfactory epithelium and retina (Fig. 5D,F; Fig. S4) and indicated selective infection of

243 neuronal cells in these tissues. Notably, even though abundant route-independent infection of the olfactory bulb

244 was detectable, presence of fewer RABV-infected cells after i.m. inoculation as compared to the i.c. route

245 demonstrated inoculation route-dependent kinetics of the centrifugal spread to peripheral nerves (Fig. 5A and 6A).

246 Similar to the infection of femoral tissues (Fig. 2 and 3), RABV P was arranged around NEFM-positive axons

247 (Fig.5J, Fig 7A-E), indicating involvement of SCs. Accordingly, RABV was detected at the convex side of the

248 myelin sheaths (Fig. 7G-J, arrowheads; Video S7). This confirmed infection of neuroglia in RABV infection of

249 peripheral nerves of the facial head region. However, in single cases RABV P was also present inside the myelin

250 sheaths, indicating RABV P accumulation in the respective axons (Fig. 7I,J, stars).

251

252

253 Discussion

254 Using organic solvent-based organ clearing [45,49] for the 3D deep tissue imaging of RABV infections [69], we

255 aimed at investigating and systematically analyzing the in vivo cycle and main checkpoints of a highly virulent

256 field RABV (rRABV Dog) on its journey through the host’s body. To this end, we provide an unprecedented

257 comprehensive overview of the local field RABV tissue tropism and spread in the late clinical phase of RABV

258 encephalitis based on high-volume immunofluorescence imaging. By analyzing peripheral tissues after both i.m.

259 and i.c. infection, we were not only able to visualize RABV infection in PNS and CNS neurons after centripetal

260 and centrifugal spread but also provide clear evidence for the infection of non-neuronal myelinating SCs in

261 peripheral nerves of hind legs and facial head regions (Fig. 2, 3 and 7). SCs constitute a glial cell type in the PNS

262 that functionally correlates to myelinating oligodendrocytes in the CNS (reviewed in [20]). Another important

263 finding is the corroboration of anterograde transport and spread of RABV in axons of peripheral nerves by infection

264 of the SCs after centrifugal spread from the CNS.

265 Myelinating SCs are peripheral neuroglia, which form the myelin sheath around axons of motor and sensory

266 neurons and, like oligodendrocytes in the CNS, are involved in saltatory conduction and trophic support for

267 neurons (reviewed in [51]). In peripheral nerves, RABV infection was associated with axon demyelination

268 [37,38,57,14,55]. T-lymphocyte-dependent immune pathogenesis was identified to be causative for RABV-

269 mediated neurotic paralysis, the disintegration of myelin sheaths, and the degeneration of axons [65]. Furthermore,

270 there has been speculation that involvement of rabies-induced neuropathogenesis in peripheral nerves results in

271 the typical rabies sings of muscle spasms and deglutition [37].

272 Although there are indications that SCs are somehow affected by RABV infections [14,37,38,55,57], evidence for

273 direct RABV infection of SCs in the PNS is lacking. While released RABV particles were detected at nodes of

274 Ranvier (known as myelin-sheath gaps occurring along a myelinated axon [40]) and between axonal and SC

275 plasma membranes [31,41,42]) only once infection of SCs is described [4]. Others failed to detect RABV infected

276 SCs (reviewed in [50]). Possible explanations for that may be the use of ultrathin sections in electron microscopy

277 in previous studies that run the inherent risk of missing rare events, RABV strain-specific differences in

278 accumulation in peripheral axons and SCs, and variations in the clinical phase of the analyzed animals.

279 SCs are immunocompetent and exert key functions in immune regulation by antigen presentation, pathogen

280 detection, and cytokine production [24,68]. Accordingly, productive or non-productive infection of these cells

281 could be critically important to immediate innate responses, local inflammatory processes, and the development

282 of adaptive immunity. Recently, we demonstrated that field RABVs, including the recombinant clone rRABV Dog

283 used in this study, are able to infect astroglia in the CNS to a remarkable degree comparable to neuron infections. 11

284 Whereas infection of astrocytes seems to be a hallmark of field RABVs, more attenuated lab strains are

285 characterized by highly restricted or absent infection of astrocytes. Accordingly, establishment of astroglia

286 infection by field RABVs was hypothesized to limit or block respective immune responses, subsequent virus

287 elimination, and delay immune pathology [48]. RABV infection of SCs in the PNS, as demonstrated in our study

288 (Figs. 2, 3 and 7), seems to represent a direct correlate to astroglia infection in the CNS. Comparable to the latter,

289 field RABV replication in peripheral neuroglia may result in local suppression of glia-mediated innate immune

290 responses and, thus, gain ability to replicate in SCs. In contrast, similar to the astroglia infection in the brain

291 [46,48], less virulent RABVs may infect SCs in an abortive manner because of a strong type I interferon response.

292 Evasion of innate and intrinsic antiviral pathways have been attributed to the RABV nucleoprotein (N), P, and

293 matrix (M) proteins [10,9,8,36,62]. As discussed above, there is reason to believe that, similar to current

294 hypotheses about the role of neuroglia infection in the CNS, virus variants are not or only inefficiently able to

295 establish infection in peripheral neuroglia. This may induce stronger local interferon responses, more severe local

296 immune pathogenesis, reduced virus replication, and damage of infected neurons. While we could even confirm

297 infection of SCs by another, bat-associated field RABV (Fig. S5), analyses with less virulent lab-adapted strains

298 and live-attenuated vaccine viruses have to be subject of further studies to assess whether the virulence-dependent

299 difference in glial cell infection observed in the brain [48] can be transferred to the SC infections in the PNS

300 described in this study.

301 Virus infection of peripheral SCs in hind legs and facial head regions by centrifugal spread after i.c. inoculation

302 (Figs. 3 and 7) as well as infection of sensory neurons in the olfactory epithelium and other tissues (Figs. 5 and 6)

303 provide evidence for anterograde transport of RABV along axons in vivo, something that has been attributed to

304 late phases of infection [16]. The long distance from the neuron soma in the respective ganglia and the tight spatial

305 association of the infected neuroglia with the axons makes it exceedingly unlikely that virus release at cell bodies

306 and extra-neuronal spread is responsible for distal SC infections.

307 Both in vitro and in vivo virus tracking already demonstrated that anterograde RABV transport through axons of

308 peripheral DRG neurons and other peripheral neurons is possible [3,60,6]. However, anterograde virus spread was

309 discussed to occur only in a passive, kinesin-independent manner and the release of infectious virus was called

310 into question [61]. Fast, post-replicative, and glycoprotein (G)-dependent anterograde axonal transport of RABV

311 in cultured DRG neurons, however, provided evidence for active transport to distal axon sites [6]. One reason for

312 the discrepancy observed between in vitro tracking and in vivo trans-neuronal tracing approaches could be the role

313 of peripheral neuroglia as major innate immune mediators in infected nerves as discussed earlier. Since the

314 standard neuronal tracer virus CVS-11 is less effective in infecting astrocytes in the brain [48], a strong local 12

315 antiviral immune response at peripheral sites may hinder CVS-11 replication in neuroglia target cells. Moreover,

316 anterogradely transported virus particles in axons may remain below the limit of detection in conventional in vivo

317 tracing approaches. Accordingly, anterograde transport in CVS-11 infection may have been overlooked because

318 peripheral neuroglia, which were used here as an indicator for anterograde spread of the used field RABV, might

319 not or only get abortively infected.

320 Beyond RABV spread in peripheral hind leg nerves after i.m. and i.c. infection, a more generalized roadmap of

321 RABV infection in the CNS and PNS could be build. As for human [58,32] and numerous animal samples (e.g.

322 [34,29,28]), abundant RABV specific signals in the spinal cord and brain could be observed (Fig. 4 and Fig. S6).

323 Virus detection in the orbital, nasal, and oral cavity confirmed previous reports about presence of RABV and

324 related lyssaviruses in a variety of other tissues [17,30,22,1], including retinal ganglion cells, their axons, and

325 lacrimal glands [25,15]. Considering the latter, a potential role of lacrimal glands in virus secretion has been

326 discussed [15]. Whereas lacrimal fluid may not contribute to the transmission of infectious virus to new hosts via

327 saliva, multiple infected sensory neurons in the olfactory and tongue epithelia (Fig. 5 and 6) with a direct

328 connection to the nasopharynx raise the question whether virus secretion at these sites could contribute to the total

329 amount of infectious virus in the salvia. For bat lyssaviruses, for instance, viral antigen was also detected in taste

330 buds of experimentally [22] and naturally infected animals [52].

331 Conclusions

332 By providing a comprehensive and detailed overview of RABV distribution in peripheral tissues after natural (i.m.)

333 and artificial (i.c.) routes of inoculation, we highlight novel insights in the cell tropism and in vivo spread of field

334 RABV. Importantly, by unveiling the infection of peripheral neuroglia, we suggest a model in which field RABV

335 infection of immunocompetent SCs is critically important for local innate immune responses in peripheral nerves.

336 This may not only contribute to the delayed immune detection of field RABVs through RABV mediated innate

337 immune response suppression in field virus infected SCs, but may also may explain the high neuroinvasive

338 potential of field RABVs when compared with less virulent lab virus strains, and manifestation of clinical

339 symptoms observed in infected animals and humans. Moreover, by demonstrating infection of myelinating SCs

340 after centrifugal spread from infected brains, we provide evidence for anterograde spread of infectious RABV

341 through axons of the peripheral nerve systems in clinical phases of rabies encephalitis.

342

343

344 Abbrevitations

345 BA: benzyl alcohol

346 BB: benzyl benzoate

347 CNS: central nervous system

348 DPE: diphenyl ether.

349 DRG: dorsal root ganglion

350 EDTA: ethylenediaminetetraacetic acid

351 G: glycoprotein

352 i.m.: intramuscular

353 i.c .: intracranial

354 LALFF M-V: State Office for Agriculture, Food Safety, and Fishery in Mecklenburg-Western Pomerania

355 M: matrix protein

356 MBP: marker myelin basic protein

357 N: nucleoprotein

358 NEFM: neurofilamten M

359 P: phosphoprotein

360 PNS: peripheral nervous system

361 RABV: rabies virus

362 PFA: paraformaldehyde

363 RNPs: ribonucleoproteins

364 SC: Schwann cell

365 TBA: tert-butanol

366 Declarations

367 Ethics Approval

368 No human material was used in this study. Animal trials have been evaluated by the ethics committee of the State

369 Office for Agriculture, Food Safety, and Fishery in Mecklenburg-Western Pomerania (LALFF M-V) and gained

370 approval with permission 7221.3-2-047/19 and 7221.3-2.1-001/18.

371

372 Acknowledgements

373 We thank Angela Hillner and Katrin Giesow for technical assistance.

374 14

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550

551 552 Fig. 1 Overview of field RABV infection in mouse hind leg cross sections after i.m. infection (9 days post- 553 inoculation). (A) Maximum z-projection of light sheet overview of a hind leg cross section [1.6x magnification; z 554 = 1,156 µm]. Red: RABV P; blue: nuclei. Green fluorescence for NEFM not shown as separation from green 555 autofluorescence was not possible at low resolution. Scale bar: 1,500 µm. (B) 3D projection of (A). (C) Maximum 556 z-projection of detail (white box in A) with infected nerve [12.6x magnification; z = 574 µm]. Scale bar: 200 µm.

557 (D) Maximum z-projection of confocal z-stack of indicated region in (C) [z = 48 µm] of infected nerve with NEFM 558 signal (green). Scale bar: 100 µm. Note: due to the separate image acquisition, the orientation of the shown nerve 559 is different to (C). (E) Maximum z-projection of detail from (D) [z = 10 µm]. Scale bar: 10 µm. (F-J) Maximum 560 z-projections of serial thick hind leg sections from the upper leg (F) to the knee (J) [1.6x magnification, z = 1,156 561 µm (F), 1,402 µm (G), 1,172 µm (H), 1,990 µm (I), 1,372 µm (J)]. For reference, see indicated slices in Fig. S2A. 562 For improved intelligibility, only RABV P (red) and nuclei (blue) are shown. Scale bar: 1,500 µm. 563

564 23

565 Fig. 2 Detection of RABV P in non-neuronal Schwann cells of hind leg tissue after i.m. inoculation (9 days post- 566 inoculation). (A,B) Maximum z-projection of high-resolution confocal z-stacks of RABV-infected nerve fiber (see 567 also Fig. 1D) in hind leg sections [z = 45 µm; Scale bar: 100 µm] (A) and detail [z = 15 µm; Scale bar: 10 µm] 568 (B). Red: RABV P; green: NEFM; blue: cell nuclei. (C,D) Volumetric 3D projections of (B) with different viewing 569 angles. The light blue box indicates the clipping plane. Arrows: RABV P surrounding axonal NEFM. (E,F) 570 Maximum z-projection of a hind leg section (E) [z = 134 µm; Scale bar: 200 µm] and detail (F) [z = 20 µm; Scale 571 bar: 10 µm] stained for RABV P (red), MBP (green) and nuclei (blue). (G,H) Volumetric 3D projections of 572 different viewing angles of (F). Arrowhead: RABV-infected Schwann cell (I-K) Single slices with RABV P (red), 573 MBP (green) and nuclei (blue). Arrowheads indicate myelin sheath/nucleus surrounded by RABV P. Scale bar: 5 574 µm. 575

576 25

577 Fig. 3 Anterograde virus spread: RABV in Schwann cells of a hind leg nerve after i.c. inoculation. (A,B) 578 Maximum z-projection of light sheet overview (A) and detail (B) of hind leg section after staining for RABV P 579 (red), and nuclei (blue). Green fluorescence for NEFM is not shown as separation from green autofluorescence 580 was not possible at low resolution. (A) Magnification of 1.6x [z = 1,944 µm; Scale bar: 1,500 µm] and (B) 581 magnification of 12.6x [z = 58 µm; Scale bar: 200 µm]. (C) Maximum z-projection of confocal z-stack [z = 37 582 µm] of infected nerve fiber from (B), now including NEFM staining (green). Scale bar: 100 µm. (D,E) Maximum 583 z-projection of details from (C) (see white boxes) [z = 5 µm (D,E)]. Scale bar: 10 µm. (F,G) Maximum z-projection 584 of hind leg nerve section stained for RABV P (red), MBP (green) and NEFM (blue) [z = 15 µm]. To visualize low 585 amounts of viral antigen, the MBP channel is excluded in (G). Scale bar: 50 µm. (H) 3D projection of detail from 586 (F) (see white box). (I-K) Single planes from detail of (F). The arrowhead indicates an RABV-infected cell. Scale 587 bar: 5 µm. 588

589 590 Fig. 4 Spinal column infection after i.m. and i.c. infection. (A,B) Maximum z-projections of light sheet 591 overviews of cross sections of the spinal column after i.m. (A) and i.c. (B) infection [2.0x magnification; z = 824 592 µm (A), 1,480 µm (B)]. Red: RABV P; blue: nuclei. Green fluorescence for NEFM not shown as separation from 593 green autofluorescence was not possible at low resolution. Stars: grey matter, triangles: white matter. Scale bar: 594 1,500 µm. (C) Maximum z-projection of detail from (A), including NEFM signals (green). Scale bar: 200 µm. 595 (D,E) Maximum z-projection of confocal z-stacks of infected spinal cord (D) [z = 31 µm; Scale bar: 100 µm] and 596 detail (E) [z = 10 µm; scale bar: 10 µm]. (F) Maximum z-projection of detail from (A) with neurons projecting 597 from the spinal cord. Scale bar: 200 µm. (G,H) Maximum z-projection of confocal z-stacks (G) [z = 36 µm, Scale 598 bar: 100 µm] and detail (H) [z = 10 µm; Scale bar: 10 µm] of projecting nerves from the spinal cord. (I) Maximum 599 z-projection of detail from (B), including NEFM signals (green). Scale bar: 200 µm. (J,K) Maximum z-projection 600 of confocal z-stacks of infected spinal cord (J) [z = 61 µm; Scale bar: 100 µm] and detail (K) [z = 20 µm; scale 601 bar: 10 µm]. (L) Maximum z-projection of detail from (B), including NEFM signals (green). Scale bar: 200 µm. 602 (M,N) Maximum z-projection of confocal z-stacks of projecting nerve (M) [z = 41 µm; Scale bar: 100 µm] and 603 detail (N) [z = 20 µm; scale bar: 10 µm]. 604

605 606 Fig. 5 Coronal overview of field RABV-infected mouse head sections after i.c. inoculation. (A,B) Maximum z- 607 (A) and 3D projection (B) of mouse head sections after intracranial infection with field RABV [1.26x 608 magnification; z = 1,606 µm]. Indirect immunofluorescence staining against RABV P (red), NEFM (green), and 609 nuclei (blue). Green fluorescence for NEFM not shown as separation from green autofluorescence was not possible

610 at low resolution. Scale bar: 2,000 µm. (C, E, G, I) Maximum z-projections of details from A (white boxes) at a 611 magnification of 12.6x. Scale bar: 200 µm. (D, F, H, J) Confocal high-resolution z-stacks (corresponding to the 612 indicated regions in C, E, G, I, respectively) [z = 35 µm (D), 32 µm (F), 36 µm (H), 40µm (J); scale bar: 50 µm 613 and 15 µm in detail]. Note: due to the separate confocal image acquisition, the orientations differ from the light 614 sheet microscopy images in C, E, G, and I. (C,D) Infected eye, (E, F) nasal epithelium, (G, H) tongue, (I, J) and 615 teeth in mouse head coronal section. (K-N) Maximum z-projection of serial mouse head sections (Fig. S2B) [1.26x; 616 z = 1,780 µm (K), 1,820 µm (L), 1,606 µm (M), 1,760 µm (N); green not shown]. Scale bar: 2,000 µm. 617

618 619 Fig. 6 RABV distribution in head cross sections after i.m. inoculation. (A,B) Maximum z- (A) and 3D projection 620 (B) of coronal mouse head sections after i.m. infection with field RABV rRABV Dog [1.26x magnification; z = 621 4,210 µm]. Indirect immunofluorescence staining against RABV P (red), NEFM (green), and nuclei (blue) 622 revealed a markedly reduced degree of infection after i.m. infection in comparison to i.c. infection (see Fig. 6). (C, 623 E, G, I) Maximum z-projections of details from A (white boxes) [12.6x; magnification] (Scale bar: 200 µm). (D, 624 F, H, J) Confocal high-resolution z-stacks (corresponding to C, E, G, I, respectively) [z = 80 µm (D), 26 µm (E), 625 72 µm (H), 74 µm (J); scale bar: 100 µm] with details (scale bar: 15 µm). (C,D) infected eye, (E, F) nasal 626 epithelium, (G, H) tongue, (I, J) and teeth in mouse head coronal section. 627

628 629 Fig 7 RABV P in axons and infected Schwann cells in coronal head sections. (A) Maximum z-projection of high- 630 resolution confocal z-stacks of mouse head sections after i.c. inoculation and staining for RABV P (red), NEFM 631 (green) and nuclei (blue) [z = 38 µm]. Arrows indicate RABV P surrounding NEFM. Scale bar: 100 µm. (B-E) 632 Maximum z-projections (B, D) and corresponding 3D projections (C, E) of details of (A) (white boxes). (F) 633 Maximum z-projection of confocal z-stacks of mouse head sections after i.c. inoculation stained for RABV P (red), 634 MBP (green) and nuclei (blue) [z = 29 µm]. Scale bar: 25 µm. (G-I) Single slices of (F). Arrowhead: RABV P 635 surrounding MBP-positive myelin sheath. Star: RABV P surrounded by MBP-positive myelin sheath. Scale bar: 636 5 µm. (J) 3D projection of (G-H). 637

638 Supplemantary Material

639 Supplementary Figures_S1-S6.pdf (including figure captions and descriptions)

640 Captions Videos S1-S7 (Captions and descriptions for supplementary videos)

641 Video S1.mp4 31

642 Video S2 reduced.avi

643 Video S3.mp4

644 Video S4.mp4

645 Video S5.mp4

646 Video S6.mp4

647 Video S7.avi

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